OBJECTIVE: The goals of this project are to identify the roles of endogenous NAD(P)H oxidases in the proliferative responses and enhanced contractility leading to airways smooth muscle (AWSM) remodeling and hyperreactivity in asthma. HYPOTHESIS: The inclusive hypothesis to be tested is that airway smooth muscle NAD(P)H oxidases are highly regulated ROS producing enzymes that play distinct roles in initiating AWSM proliferation and contraction. SPECIFIC AlMS: The first specific aim will characterize the NAD(P)H oxidase(s) of airway smooth muscle (AWSM) cells and determine its contribution to the generation of ROS. This aim will address the hypothesis that NAD(P)H Oxidase 4 (Nox4) bound to membranes in the endoplasmic reticulum is the catalytic subunit of the NAD(P)H oxidase in proliferating AWSM. The second specific aim will define the role of NAD(P)H oxidase(s) in AWSM proliferation. This aim will test the hypotheses that specific growth factors induce AWSM proliferation via activation of the Nox4 oxidase with resultant transactivation of nuclear factor-kappa B (NF-kB). The third specific aim will define the role of NAD(P)H oxidase(s) in AWSM contractile function. This aim will test the hypothesis that ROS generated by an NAD(P)H oxidase induce AWSM contraction, but that components of the oxidase in the contractile phenotype are distinct from those of the proliferative phenotype. RESEARCH PLAN: For the first aim, our strategy is to define and fully characterize the structure of the components responsible for AWSM NAD(P)H oxidase activity and pinpoint their subcellular location and interaction. We will also characterize the activity, the redox midpoint potential of AWSM NAD(P)H oxidase and the role of individual components in the generation of ROS. For the second aim we will utilize purified cells with deficiencies of NAD(P)H oxidase components to directly establish the importance of the oxidase in AWSM proliferation. Emphasis will be placed on establishing the role of the oxidase in transactivation of NF-kB, a factor essential for smooth muscle proliferation. For the third aim we will again utilize purified cells and animals with genetic deficiences of NAD(P)H oxidase components to directly establish the importance and characteristics of the oxidase that regulates airways contractile function. Emphasis will be placed upon investigating whether there is a phenotype switch in the oxidase components. SIGNIFICANCE: The work will provide a better understanding of the importance of AWSM NAD(P)H oxidase in mediating airways smooth muscle remodeling in asthma and contractility in the asthmatic state, with broad importance in the role of ROS in respiratory physiology and disease.